Two new dimethylpyranoflavanones from the roots of Melodorum fruticosum

Abstract Two previously unreported dimethylpyranoflavanones, pyronomelodorones A and B (1 and 2), along with five known compounds, 7-O-methyldihydrowogonin (3), 5,6,7-trimethoxyflavanone (4), 5,6-dihydroxy-7-methoxy-dihydroflavone (5), 5,7,8-trimethoxydihydroflavone (6), and pinostrobin (7), were isolated from the roots of Melodorum fruticosum. The structures of all isolates were fully characterized using spectroscopic data and comparison with the previous literature. All isolates were evaluated for their in vitro α-glucosidase inhibition and their cytotoxicity against KB, HepG2, and MCF7 cell lines. Among the isolates, compound 1 exhibited the most inhibitory activity against α-glucosidase and was superior to the positive control with an IC50 value of 1.32 μM. Compounds 1 and 2 showed weak cytotoxicity against the three human cancer cell lines, with IC50 values in the range of 53.3–79.0 μM.

The dimethylpyrano unit at C-6 (d C 133.4) and C-7 (d C 128.0) was substantiated by the observation of the HMBC correlations from the cis-coupled olefin proton signal at d H 6.58 (d, J ¼ 10.0 Hz, H-1 00 ) to C-3 00 (d C 74.7), C-6, and C-8 (d C 114.3).The location of the prenyl unit at C-8 was confirmed by the HMBC correlations from H-1 000 (d H 3.27, m) to C-7, C-8, C-9 (d C 151.4), and C-2 000 (d C 122.6) (Figure S1).Furthermore, the long-range HMBC cross peak from the hydroxyl group (d H 11.62, s, 5-OH) at C-5 (d C 147.3) to the carbon signals at d C 133.4 (C-6) and 107.6 (C-10) suggested that the location of the remaining oxygenated aromatic carbon be at C-6, which must be the cyclization site of the dimethylpyrano unit.This unusual phenomenon implied that 1 had the same fusion pattern for the dimethylpyrano unit ring on the A ring as flavanone derivatives isolated from Eriosema tuberosum (Ma et al. 1995), which is very rarely found in natural products.The ring B protons appeared as five proton multiplets in the d H 7.38-7.52ppm region, which was typical of the mono-substituted ring B and that is commonly found in flavanones (Zheoat et al. 2021).Besides ring B protons, no other aromatic protons were found on the 1 H NMR spectrum, indicating that ring A was fully substituted.The spectroscopic data of 1 were similar to those reported for mundulin (Alavez-Solano et al. 2000), except for the presence of the oxygenated aromatic carbon at C-6 and the arrangement of the dimethylpyrano unit in 1 (Figure 1).The absolute configuration of C-2 was determined as R-configuration through contrastive analysis of the specific optical rotation data of 1 ([a] D þ4.7) with the known compounds, (2 R)-6formyl-5,7-dihydroxyflavanone ([a] D þ31) and (2 R)-8-formyl 5,7-dihydroxyflavanone ([a] D þ31) (Zaki et al. 2016), whose absolute configuration was established via total synthesis and the electronic circular dichroism (ECD) spectroscopic data.Thus, the structure of 1 was determined as shown, and this compound has been given the trivial name as pyronomelodorone A.
Compound 2 was obtained as pale yellow gum and optically active ½a 20 D þ9.6 (c 0.00021, CHCl 3 ).Its molecular formula, C 20 H 18 O 4 , was determined from the HRESIMS with m/z 323.1278 ) and confirmed by its NMR spectral data (Table S1).This was further confirmed by the 1 H and 13 C NMR spectral data, which disclosed two methyl, one methylene, one oxymethine, two cis-olefinic methine, six aromatic methine, and eight quaternary carbons.It was clear from the 13 C NMR chemical shift at d C 42.8 (C-3), 78.3 (C-2), and 198.6 (C-4) that 2 was a flavanones (Jenkins et al. 1999).The NMR (Table S1) and specific optical rotation ([a] D þ9.6) data of 2 were found to be very similar to those of 1 except for the absence of a prenyl substituent at C-8.This was confirmed by the HMBC correlations of H-8 (d H 6. All isolates obtained were evaluated for their a-glucosidase inhibitory activity, with acarbose used as a reference compound.The resulting IC 50 values, as listed in Table S4, indicated that compounds 1-4 and 7 showed significant a-glucosidase inhibitory activity with IC 50 values ranging from 1.32 to 3.16 lM, which were more potent than acarbose (IC 50 ¼ 179 lM).Of these, compound 1 possessed the strongest potent a-glucosidase inhibitory property with an IC 50 value of 1.32 lM.On the other hand, compounds 5 and 6 displayed no a-glucosidase inhibitory activity (IC 50 >256 lM).The results suggested that compounds 1-4 and 7 were active a-glucosidase inhibitors that could be used as effective hypoglycemic agents for diabetes therapy.
All isolates were also assessed for their in vitro cytotoxicity against KB, HepG2, and MCF7 cell lines, and the IC 50 values are summarized in Table S5.Compounds 1-7 exhibited different degrees of cytotoxicity toward three human cancer cell lines.Among them, only compounds 1 and 2 displayed weak cytotoxicity against three human cancer cells with IC 50 values in the range of 53.3-79.0lM, while the other compounds exhibited no cytotoxic activity (IC 50 >100 lM).

General experimental procedures
Optical rotations were measured on a A.KR € USS Optronic P8000 polarimeter (KR € USS, Hamburg, Germany).IR spectra were obtained with a Jasco 6600 FT-IR spectrometer using an ATR technique (Jasco, Japan).The NMR experiments were acquired on a Bruker 600 AvanceNEO spectrometer and a Bruker Avance 500 III in DMSO-d 6 (Merck, Darmstadt, Germany).HRESIMS spectra were recorded using a X500 R QTOF model mass spectrometer (Sciex, USA) and Dionex Ultimate 3000 HPLC system hyphenated with a Q Exactive Hybrid Quadrupole Orbitrap MS (Thermo Fisher Scientific).UV-visible absorption spectra obtained with a UV-2550 UV-vis spectrometer (Shimadzu).Silica gel 60 (0.040-0.063 mm, Silicycle) and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) were used for column chromatography.

a-Glucosidase inhibitory assay
All tested isolates and the positive control (acarbose, Bayer Vitol Leverkusen, Germany) were subjected to a-glucosidase inhibitory evaluation employing the colorimetric method (Sichaem et al. 2017).Briefly, 0.2 U/mL of a-glucosidase (Saccharomyces cerevisiae, Sigma-Aldrich, St. Louis, USA) and substrate (5.0 mM p-nitrophenyl-a-D-glucopyranoside, Sigma-Aldrich) were dissolved in 100 mM pH 6.9 sodium phosphate buffer.The inhibitor (50 lL) was preincubated with a-glucosidase at 37 C for 20 min, and then the substrate (40 lL) was added to the reaction mixture.The enzymatic reaction was carried out at 37 C for 20 min and stopped by adding 0.2 M Na 2 CO 3 (130 lL).Enzymatic activity was quantified by measuring absorbance at 405 nm.All samples were analyzed in triplicate at five different concentrations around the IC 50 values, and the mean values were retained.The inhibition percentage (%) was calculated by the following equation: Inhibition (%) ¼ [1 -(A sample /A control )] Â 100.

Cytotoxic assay
All tested isolates and the positive control were subjected to in vitro cytotoxic evaluation against human epidermoid carcinoma (KB), human hepatocellular carcinoma (HepG2), and human breast adenocarcinoma (MCF7) cell lines employing the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St. Louis, USA) surrogate assay as previously reported (Kongkathip et al. 2003).The cell lines used in the study were purchased from ATCC.The positive control was ellipticine (Sigma-Aldrich), a powerful anticancer medication with various modes of action.The cancer cells were grown in Dulbecco's Modified Eagle's Medium (Gibco, Rockville, USA) at 37 C in a 5% CO 2 environment with 10% fetal bovine serum (FBS, Sigma-Aldrich), 1% penicillin and streptomycin (Sigma-Aldrich), and 1% L-glutamine (Sigma-Aldrich).By dissolving the compounds under investigation in DMSO (20 mg/mL), the compounds were added at concentrations ranging from 0.5 to 128 lg/mL, and the incubation was carried out once more for 72 h under the same circumstances.Following the procedure, an MTT solution (10 lL, 5 mg/mL) was added to each well and further incubated for 3 h.After centrifugation at 1400 rpm for 5 min at 4 C, the supernatant was decanted and DMSO (100 mL/well) was added to allow formazan product solubilization, which was subsequently measured by a microplate reader (Tecan Trading AG, Switzerland) at wavelength 550 nm.The percentage of cell viability
36, s) to C-6 (d C 133.2), C-9 (d C 154.3), and C-10 (d C 107.8); of H-1 00 (d H 6.40, s) to C-3 00 (d C 75.7), C-7 (d C 129.6), and C-8.Based on the above data and comprehensive 2 D NMR experiments (HSQC and HMBC), the structure of 2 was unambiguously established as shown in Figure 1, and it has been accorded the trivial name as pyronomelodorone B.
3.2.Plant material M. fruticosum roots was collected in Lam Dong province, Vietnam in July 2017.The material was authenticated by Dr. Dang Van Son-Institute of Tropical Biology.A voucher specimen (No US-A012) was deposited at the Herbarium of the Department of Organic Chemistry, Faculty of Chemistry, University of Science, National University-Ho Chi Minh City, Vietnam.